ABSTRACT Bladder cancer kills 12,000 Americans each year, but few research projects are targeted to this disease. For most of these patients, the cause of death is attributable to metastatic spread, commonly to the lungs. Hence, a fuller understanding of the molecular mechanisms driving the dissemination and growth of bladder cancer to the lungs is likely to present new therapeutic opportunities. The Ras family small GTPases RalA and RalB have been shown to play important roles in human tumor formation and progression. Specifically, RalB is elevated in bladder cancer and promotes migration and metastasis. Genome-wide analysis of RalB dependent changes in gene expression revealed that RalB regulates the expression of CD24, a GPI-linked glycoprotein, necessary for the growth of many human cancer cell lines. CD24 is also a biomarker of metastasis in bladder cancer. RalB induction of CD24 expression involves the Ral effector RalBP1, which is also elevated in bladder cancer, and the zinc finger transcription factor RREB1. Our Guiding Hypothesis is that a novel RalB RalBP1 RREB1 CD24 signaling pathway that was discovered by this project, promotes bladder cancer metastasis. To test this hypothesis we propose the following Specific Aims: In Aim 1, the mechanistic contributions of RalB and RalBP1 to bladder cancer lung metastasis will be dissected using mutants of both that are impaired in specific functions and effector interactions. Since RalA does not promote migration or metastasis, we will use RalA/RalB chimeras to identify RalB sequences necessary for metastasis and subsequently proteins that specifically bind to RalB via those regions. Successful completion of this aim should result in new targets for drug discovery. Aim 2 will determine how RalB and RalBP1 regulate RREB1 activity and evaluate the requirement for RREB1 regulation in CD24 expression. Since genome-wide profiling of RalB dependent gene expression led to the discovery of the metastasis biomarker CD24, we will use a human bladder cancer tissue repository and advanced computational tools to generate a gene signature of RalB expression. Given the importance of RalB in bladder cancer progression, the ability of this signature to predict the development of metastatic disease in patients will be evaluated. Eventually, this signature may be of prognostic value and an avenue to select patients for individualized RalB targeted therapy. In Aim 3 we will evaluate the requirement for CD24 in bladder cancer metastasis. An existing CD24 knockout mouse will be used to study the role of CD24 in a chemical carcinogenesis model of bladder cancer formation, invasion, and metastasis. In a preclinical study, we will also test whether anti-CD24 immunotherapy can block the growth of established human bladder cancer lung metastases. These mechanistic studies of a novel signaling pathway will provide molecular information on how Ral GTPases promote bladder cancer lung metastasis, thus providing both a framework for future patient risk stratification and opportunities for novel therapies of metastatic disease. PROJECT NARRATIVE Bladder cancer kills 12,000 Americans each year, but few research projects are targeted to this disease. For most of these patients, the cause of death is attributable to metastatic spread, commonly to the lungs. The goal of this project is to understand the mechanisms that underlie lung metastasis in human bladder cancer and use this knowledge to predict and treat this condition in patients.